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This study shows that the product of the hoxZ gene of Alcaligenes eutrophus HI6 is a 6-type cytochrome (cytochrome bJ, which is essential for anchoring the membrane-bound hydrogenase (MBH) complex to the periplasmic side of the membrane and for H,-coupled respiration. The hoxZ product is not required for MBH translocation and H,-dependent reduction of the redox dye, 2,3,5-triphenyl-2-tetrazolium chloride. The lack of cytochrome b, does not affect the electron-transport activities linked to oxidation of succinate and NADH, although it enhances the electron-flow rate through the cytochrome-c oxidase pathway in hoxZA membranes. We show that the hoxZ product is a dihaem cytochrome b (haems with of + 10 mV and + 166 mV) involved in H,-dependent electron transfer. We conclude that cytochrome b, of the A. eutrophus MBH complex is the link necessary for transfer of electrons from H, to the ubiquinone pool and that it is required for attachment of MBH to the membrane.Keywords: cytochrome b subunit; hydrogen respiration ; hoxZ gene; membrane-bound hydrogenase; Alcaligenes eutrophus.Alcaligenes eutrophus H16, a gram-negative respiration-dependent bacterium, belongs to the group of facultative lithoautotrophs that can use hydrogen as sole energy source [l]. Oxidation of hydrogen is mediated by two [NiFeI-containing hydrogenases : a cytoplasmic, heterotetrameric NAD-reducing enzyme (SH), which consists of four subunits encoded by the genes hoxF, hoxU, hoxH and hoxY [ 2 ] ; and a membrane-bound hydrogenase (MBH), which is composed of a small and a large subunit, encoded by the genes hoxK and hoxC, respectively [3]. The structural genes for SH and MBH are arranged in two separate operons tightly clustered with sets of accessory genes that are required for the formation of enzymatically active hydrogenase. The accessory-gene products participate in a series of complex post-translational events involving metal-center assembly, C-terminal proteolytic processing, oligomerization and, in the case of the MBH, translocation [4 -61.Crystal-structure analysis of the periplasmic [NiFe] hydrogenase of Desulfovibrio gigas has shown that the binuclear metal center is deep inside the protein and that the mature large subunit is devoid of a C-terminal extension [7]. Evidence for the presence of specific proteases that remove at least 15 C-terminal residues has been documented in a variety of phylogenetically distant species, including A. eutrophus [X- nological assays revealed that cells of A. eutrophus, containing active MBH, produce the small and large subunits of the enzyme in two electrophoretically distinct conformations. It was suggested that the conversion of the two subunits into the catalytically active membrane-associated heterodimer is dependent on the function of accessory-gene products [12]. More recently, we described two classes of mutants with in-frame deletions in the eight MBH-linked accessory genes. Class-I mutants, affected in hoxM, hoxO and hoxQ, are totally devoid of MBH activity, whereas class-I1 mutants, harboring del...

Chloroform (CF) is largely produced by both anthropogenic and natural sources. It is detected in ground and surface water sources and it represents the most abundant halocarbon in the atmosphere. Microbial CF degradation occurs under both aerobic and anaerobic conditions. Apart from a few reports describing the utilization of CF as a terminal electron acceptor during growth, CF degradation was mainly reported as a cometabolic process. CF aerobic cometabolism is supported by growth on short-chain alkanes (i.e., methane, propane, butane, and hexane), aromatic hydrocarbons (i.e., toluene and phenol), and ammonia via the activity of monooxygenases (MOs) operatively divided into different families. The main factors affecting CF cometabolism are (1) the inhibition of CF degradation exerted by the growth substrate, (2) the need for reductant supply to maintain MO activity, and (3) the toxicity of CF degradation products. Under anaerobic conditions, CF degradation was mainly associated to the activity of methanogens, although some examples of CF-degrading sulfate-reducing, fermenting, and acetogenic bacteria are reported in the literature. Higher CF toxicity levels and lower degradation rates were shown by anaerobic systems in comparison to the aerobic ones. Applied physiological and genetic aspects of microbial cometabolism of CF will be presented along with bioremediation perspectives.

The wide anthropogenic use of selenium compounds represents the major source of selenium pollution worldwide, causing environmental issues and health concerns. Microbe-based strategies for metal removal/recovery have received increasing interest thanks to the association of the microbial ability to detoxify toxic metal/metalloid polluted environments with the production of nanomaterials. This study investigates the tolerance and the bioconversion of selenite (SeO) by the aerobically grown Actinomycete Rhodococcus aetherivorans BCP1 in association with its ability to produce selenium nanoparticles and nanorods (SeNPs and SeNRs). The BCP1 strain showed high tolerance towards SeO with a Minimal Inhibitory Concentration (MIC) of 500mM. The bioconversion of SeO was evaluated considering two different physiological states of the BCP1 strain, i.e. unconditioned and/or conditioned cells, which correspond to cells exposed for the first time or after re-inoculation in fresh medium to either 0.5 or 2mM of NaSeO, respectively. SeO bioconversion was higher for conditioned grown cells compared to the unconditioned ones. Selenium nanostructures appeared polydisperse and not aggregated, as detected by electron microscopy, being embedded in an organic coating likely responsible for their stability, as suggested by the physical-chemical characterization. The production of smaller and/or larger SeNPs was influenced by the initial concentration of provided precursor, which resulted in the growth of longer and/or shorter SeNRs, respectively. The strong ability to tolerate high SeO concentrations coupled with SeNP and SeNR biosynthesis highlights promising new applications of Rhodococcus aetherivorans BCP1 as cell factory to produce stable Se-nanostructures, whose suitability might be exploited for biotechnology purposes.

Tellurite (TeO3(2-)) is the most toxic and soluble oxyanion among tellurium (Te) compounds. The effects of the metalloid anion on the oxidative stress response of the obligate aerobe Pseudomonas pseudoalcaligenes KF707 were investigated. Cells treated with sub-lethal concentrations of TeO3(2-) showed neither adaptation to it nor cross-protection against oxidants such as 1,1'-4,4'-bipyridinium dichloride (paraquat, PQ2+), diazenedicarboxylic acid bis-N,N-dimethylamide (diamide), tert-butyl hydroperoxide (tBH) and hydrogen peroxide (H2O2). Notably, TeO3(2-) exerted a synergic effect on the toxicity of these latter oxidants. Tellurite was shown to decrease the cellular content of reduced thiols (RSH) with a consequent increase in the production of reactive oxygen species (ROS) and stimulation of the superoxide dismutase (SOD) activity. However, since the time course of ROS production by TeO3(2) (t1/2 > 30 min) was much slower than that with PQ2+ and/or diamide (t1/2

The ability of a Rhodococcus aetherovorans strain, BCP1, to grow on butane and to degrade chloroform in the 0-633 microM range (0-75.5 mg l(-1)) via aerobic cometabolism was investigated by means of resting-cell assays. BCP1 degraded chloroform with a complete mineralization of the organic Cl. The resulting butane and chloroform maximum specific degradation rates were equal to 118 and 22 micromol mg(protein)(-1)day(-1), respectively. Butane inhibition on chloroform degradation was satisfactorily interpreted by means of a model of competitive inhibition, with an inhibition constant equal to 38 % of the estimated butane half-saturation constant, whereas chloroform (at 11 microM) did not inhibit butane utilization. Acetylene (1,720 microM) induced an almost complete inactivation of the degradation of both butane and chloroform, indicating that the studied cometabolic process is mediated by a monooxygenase enzyme. BCP1 proved capable of degrading vinyl chloride and 1,1,2-trichloroethane, but not 1,2-trans-dichloroethylene. BCP1 could grow on the intermediates of the most common butane metabolic pathways and on the aliphatic hydrocarbons from ethane to n-heptane. After growth on n-hexane, it was able to deplete chloroform (13 microM) with a degradation rate higher than that obtained, at the same chloroform concentration, after growth on butane.

Pseudomonas pseudoalcaligenes KF707 is naturally resistant to the toxic metalloid tellurite, but the mechanisms of resistance are not known. In this study we report the isolation of a KF707 mutant (T5) with hyperresistance to tellurite. In order to characterize the bacterial response and the pathways leading to tolerance, we utilized Phenotype MicroArray technology (Biolog) and a metabolomic technique based on nuclear magnetic resonance spectroscopy. The physiological states of KF707 wild-type and T5 cells exposed to tellurite were also compared in terms of viability and reduced thiol content. Our analyses showed an extensive change in metabolism upon the addition of tellurite to KF707 cultures as well as different responses when the wild-type and T5 strains were compared. Even in the absence of tellurite, T5 cells displayed a "poised" physiological status, primed for tellurite exposure and characterized by altered intracellular levels of glutathione, branched-chain amino acids, and betaine, along with increased resistance to other toxic metals and metabolic inhibitors. We conclude that hyperresistance to tellurite in P. pseudoalcaligenes KF707 is correlated with the induction of the oxidative stress response, resistance to membrane perturbation, and reconfiguration of cellular metabolism.Genomic studies have provided a rationale for the ability of bacteria to adapt to environmental changes or adverse conditions, such as exposure to toxic metals (8). Although valuable, these investigations have shortcomings for the assignment of regulatory or metabolic functions to novel genes (23). As a consequence, transcriptomics, proteomics, and metabolomic analyses need to be undertaken, in order to measure the functional cellular units in response to genetic as well as environmental perturbations. The metabolome is defined as the quantitative collection of the low-molecular-weight molecules (metabolites) required for growth and normal function of a cell (24). On the basis of this definition, metabolomics is the characterization and quantification of the cellular metabolome and provides a snapshot of the metabolic status of a cell in a particular physiological condition (14). Nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomics, as it allows for the identification and quantification of a broad range of cellular metabolites simultaneously (15).